The goal of this project is to develop an innovative method for mechanically enhancing bone formation by lowering the mechanical threshold signal required for that formation by osteoblasts. Current clinical methods utilize approaches that do not necessarily target new formation where most needed and the drugs are subject to off-target effects. Bone has a principally mechanical function. Thus, adaptation to mechanical loading strengthens the skeleton in a site and magnitude appropriate manner, at least in healthy people. Those at risk for osteoporosis often cannot generate, without injury, the high impacts or resistive-type loads required to form adequate new bone formation. Thus, this work envisions applications to populations at risk for fracture, to allow for safe, lower levels of impact-type loading. Mechanical loads are detected by osteocyte cells via fluid flow through small tunnels buried within the bone matrix. The osteocytes are mechanical sensors and signal for new bone formation upon stimulation with mechanical loads. The first signals are immediate, regular, intermittent cytoplasmic calcium ion (Ca2+) spikes. The spikes are triggered by oscillatory fluid shear stress (OFSS) that induces channels in the osteocyte membrane to open to extracellular Ca2+ resulting in the spikes. The channels open upon stimulation with ATP, the energy molecule of the body. These signaling events are fast and very few are required to result in eventual new bone formation. However, the magnitude of formation response appears dependent on the number of osteocytes that exhibit spiking. That number depends on the magnitude of the loads induced and the concentration of ATP in the osteocyte microenvironment. ATP is produced by the osteocytes through the breakdown of glucose. Established in vitro and in vivo models will be used to test whether a drug administered immediately prior to a bout of controlled loads can lower the osteocyte threshold for response to mechanical loads and result in greater bone formation. Specific Aim 1 tests the hypothesis that increased number of osteocytes are stimulated to exhibit Ca2+ spikes and downstream responses (?-catenin nuclear translocation and expression of Wnt targets) when OFSS is applied with the drug in the medium. We will quantify the responding osteocytes at multiple levels of OFSS and multiple concentrations of the drug. Specific Aim 2 tests the hypothesis that the drug lowers the mechanical threshold for a bone formation response to a single bout of controlled in vivo loading. The primary outcomes will be the bone formation rate parameters for mineral apposition and mineralizing surface. In vivo, controlled mechanical loads will be applied to the mouse tibia that have demonstrated nearly linear increases in bone formation response with increase in mechanical load magnitude. If this concept is proven then the immediate impact of this work would be to address the unmet need to provide new bone formation where most mechanically relevant in the prevention of osteoporotic fractures. Thus, this project is significant because of the need in aged populations and in many conditions, including menopause and diabetes, to attain the mechanical signals necessary to generate bone formation responses.